1. Field of The Invention
This invention relates generally to a scavenger system which can eliminate ascorbic acid interference in a large number of liquid and dry phase assay systems, particularly assay systems based upon an oxidase-peroxidase coupled reaction such as conventional assays used to determine occult blood, cholesterol, triglycerides, uric acid and the like in body fluids. More specifically, this invention is directed to an agent which quickly and selectively oxidizes ascorbate (the ion form of ascorbic acid) in an analytical sample, such as urine, before the ascorbate is able to interfere with an assay's reagent system.
2. Discussion of the Prior Art
The most common form of ascorbic acid is typically referred to as Vitamin C. This vitamin is a vital nutrient and is found in many naturally occurring foods, such as fruits and vegetables. Vitamin C can also be synthesized and is therefore available as a food additive or in tablet form.
The health benefits of Vitamin C have been known for some time. Over a century ago, sailors discovered that they would become severely ill on long voyages unless they ate certain foods rich in this vitamin. Recent medical studies have suggested that large doses of Vitamin C can be therapeutic in treating the common cold. As a result, Vitamin C is a relatively popular nutrient and is therefore a popular food additive and a popular component of vitamin pills and the like.
However, a person's body will generally absorb Vitamin C only to the extent necessary to meet the body's short term needs. The vitamin is generally not stored within the body, and excess Vitamin C is typically disposed of by means of the body's urinary system. As a result, Vitamin C is commonly found in urine samples used in medical analysis and the like.
Urine analysis is an important part of modern health care, and substantial knowledge concerning a patient's physiological condition can be obtained by monitoring urine components such as glucose, occult blood, cholesterol, triglycerides, or uric acid. However, many commonly known urine assay systems are adversely affected to some degree by ascorbic acid.
Ascorbate is a reductant which can interfere with an assay's reagent system. Many assays have redox indicator systems which will change color as the indicator changes from a reduced to an oxidized state due to the presence of an oxidizer. In a typical assay, the reagent system is designed to cause redox indicator oxidation in proportion to the amount of analyte in a sample. As a reductant, ascorbate can interfere with such systems.
However, ascorbate can be oxidized and if ascorbate is oxidized before it interferes with an assay system the ascorbate will not be able to act as a reductant and therefore will not cause interferance.
A number of systems are known which are directed to the oxidation of ascorbate. Pecht, I., et al., "The Copper-Poly-L-Histidine Complex: I. The Environmental Effect of the Polyelectrolyte on the Oxidase Activity of Copper Ions", J. Am. Chem. Soc., 89:1587, (1968) shows that ascorbate can be oxidized by means of oxygen and a copper catalyst. Also, polyhistidine (PLH) is shown to enhance the catalytic efficiency of copper II towards negatively-charged and neutral substrates such as ascorbate.
Vengerova, N.A., Kirsh, Y.E. and Kabanov, V.A., "The ascorbate-oxidase activity of the Cu.sup.+2 -poly-4-vinylpyridine complex alkylated with bromoacetic acid." Vysokomol. soyed., A 13, No. 11, pp. 2509-2517 (1971) (translated by K.A. Allen) shows a method of synthesizing carboxymethyl derivatives of poly-4-vinyl-pyridine and shows that a Cu II polymer complex will increase ascorbate oxidizing activity relative to copper ions alone.
It is also known that a histamine-latex can be synthesized and added to CuSO.sub.4 to make the complex-latex-histamine-Cu(II). The complex behaves as a Michaelis-Menten catalyst and has been found to be stable and reusable as a catalyst for the oxidation of ascorbic acid. See generally, Sun, Z., Jan, C., and Ketano, H., "Studies on Functional Latices: Catalytic Effects of Histamine-containing Polymer-latex-copper (II) Complex on the Oxidation of Ascorbic Acid." Macromolecules, 19:984-987 (1986).
It is also known that ascorbic acid can be oxidized with molecular oxygen catalyzed by a complex of copper II and poly-4-vinyl-pyridine. The mechanism involves the interaction of copper II with the ascorbate anion to yield copper I ions. Another stage of the catalytic effect is the oxidation of copper I by molecular oxygen. See generally, Skurlator, Y.I. et al., "The Mechanism of Ascorbic Acid Oxidation by Cu(II)-poly-4-vinyl-Pyridine Complexes." European Polymer Journal, 15:811-815 (1979).
Also, European Patent Application 0037056 describes the use of iodate in diagnostic methods to avoid interference by reducing agents, including ascorbic acid. U.S. Pat. No. 4,288,541, by Magers, "Ascorbate Resistant Composition Test Device and Method for Detecting a Component in Liquid Test Sample", discloses the use of mercuric ion complex as a scavenger system for the removal of ascorbic acid in a test device for urinary glucose.
Many of the above references relate to the oxidation of ascorbate by reducing Cu.sup.+2 to Cu.sup.+1. However, Cu.sup.+1 is also a reductant and therefore will interfere with an assay in a manner similar to nonoxidized ascorbate. The Vengerova reference re-oxidizes Cu.sup.+1 to Cu.sup.+2 using oxygen. However, oxygen can be a very inconvenient reagent, since it is a gas at room temperature. Furthermore, all of the ascorbate oxidizing substances discussed in the above-mentioned articles are insoluble in water and organic solvents. As such, it would be very difficult to apply the substances as a uniform coating on film and therefore they would not be suitable for a dry reagent system, such as a conventional dip-and-read reagent test strip system. Furthermore, the prior art substances do not have sufficient reactivity to remove (scavenge) ascorbate at a rate sufficient to prevent unwanted interference with a conventional redox indicator system.
Consequently, it is an object of the present invention to provide an agent which can be used in an assay system to oxidize (scavenge) ascorbate at a rate sufficient to prevent unwanted ascorbate interference.
A further object of this invention is to provide an ascorbate scavenger system which can be incorporated into a conventional "dip-and-read" reagent test strip.
Other objects and features of the present invention will become obvious to those of ordinary skill in the art upon reading the following specification.